The invention relates to genetically modifying plants, e.g., trees, through manipulation of the lignin biosynthesis pathway, and more particularly, to genetically modifying plants through the down regulation of 4-coumarate Co-enzyme A ligase (4CL) to achieve faster growth. Down regulation of 4CL may also achieve altered lignin content, and/or altered lignin structure, and/or altered cellulose content, and/or altered disease resistance of the trees. Moreover, promoters of the 4CL genes are useful to drive gene expression specifically in xylem tissue or specifically in epidermal tissues.
Genetic engineering of plants to conform to desired traits has shifted the emphasis in plant improvement away from the traditional breeding programs during the past decade. Although research on genetic engineering of plants has been vigorous, the progress has been slow.
The ability to make plants grow faster continues to be the top objective of many companies worldwide. The ability to genetically increase the optimal growth of plants would be a commercially significant improvement. Faster growing plants could be used by all sectors of the agriculture and forest products industries worldwide.
Lignin, a complex phenolic polymer, is a major component in cell walls of secondary xylem. In general, lignin constitutes 25% of the dry weight of the wood, making it the second most abundant organic compound on earth after cellulose. Although lignin plays an important role in plants, it usually represents an obstacle to utilizing biomass in several applications. For example, in wood pulp production, lignin has to be removed through expensive and polluting processes in order to recover cellulose.
Thus, it is desirable to genetically engineer plants with reduced lignin content and/or altered lignin composition that can be utilized more efficiently. Plants that could be genetically engineered with a reduced amount of lignin would be commercially valuable. These genetically engineered plants would be less expensive to pulp because, in essence, part of the pulping has already been performed due to the reduced amount of lignin. Further, plants with increased cellulose content would also be commercially valuable to the pulp and paper industry.
Disease resistance in plants is also a desirable plant trait. The impact of disease resistance in plants on the economy of plant products industry worldwide is significant.
Thus, what is needed is the identification and characterization of genes useful to enhance plant growth, alter lignin content and/or structure in plants, alter cellulose content in plants, and/or provide or enhance disease resistance of plants.
The invention provides a method to genetically alter plants through the down regulation (decrease) or inhibition of native (endogenous) 4-coumarate Co-enzyme A ligase (4CL) in that plant. Such down regulation of 4-coumarate Co-enzyme A ligase results in faster growth, and/or reduced lignin content, and/or altered lignin structure, and/or altered cellulose content, and/or altered disease resistance in the genetically altered plant. The invention also provides for genetically engineered plants, e.g., transformed or transgenic plants, which have been altered to down regulate or inhibit native 4-coumarate Co-enzyme A ligase in the plant so as to achieve faster growth, and/or reduced lignin content, and/or altered lignin structure, and/or increased cellulose content, and/or increased disease resistance. Preferred genetically altered plants include trees, e.g., angiosperms or gymnosperms, forage crops, and more preferably a forest tree, e.g., Populus. Preferred angiosperms include, but are not limited to, Populus, Acacia, Sweetgum, yellow poplar, maple and birch, including pure lines and hybrids thereof. Preferred gymosperms include, but are not limited to, Pine, Spruce, Douglas-fir and hemlock.
The invention further provides a transgenic plant, the genome of which is augmented by a recombinant DNA molecule encoding 4-coumarate Co-enzyme A ligase, or a recombinant DNA molecule comprising an antisense 4-coumarate Co-enzyme A ligase gene, or a fragment thereof. The recombinant DNA molecule is expressed so as to down regulate, decrease or inhibit lignin pathway 4-coumarate Co-enzyme A ligase.
The invention also provides an isolated and purified DNA molecule comprising a DNA segment comprising a transcriptional regulatory control to region of a 4-coumarate Co-enzyme A ligase gene. Preferably, the transcriptional regulatory region comprises a promoter. Tissue specific promoters of a 4-coumarate Co-enzyme A ligase gene can be used to manipulate gene expression in target tissue such as xylem and epidermal tissues, as described hereinbelow. Preferably, the promoter is derived from aspen DNA. Therefore, the invention also provides an expression cassette comprising a transcriptional regulatory region of a 4-coumarate co-enzyme A ligase gene, a method of using the region to express a preselected DNA segment in a tissue-specific manner in plant cells, and a transgenic plant comprising the expression cassette.
Also provided is a method to alter, e.g., enhance, plant growth. The method comprises introducing an expression cassette into cells of a plant, e.g., the cells of a tree, so as to yield genetically altered plant cells. The expression cassette comprises a recombinant DNA molecule, segment, or sequence, comprising a 4-coumarate Co-enzyme A ligase gene, or a fragment thereof. Preferably, the 4-coumarate Co-enzyme A ligase gene, or fragment thereof, is in antisense orientation. The 4-coumarate Co-enzyme A ligase gene may be a homologous or heterologous 4-coumarate Co-enzyme A ligase gene. The transformed plant cells are regenerated to provide a genetically altered, e.g., transgenic, plant. The recombinant DNA is expressed in the cells of the regenerated, genetically altered plant in an amount that confers enhanced or accelerated growth to the regenerated, genetically altered plant relative to the corresponding non-genetically altered plant. Preferably, the genetically altered plant is a tree. It is preferred that a genetically altered tree of the invention has an increase in height, leaf size, diameter and/or average internode length relative to the corresponding non-genetically altered tree.
Hence, the invention also provides for a genetically altered plant, the genome of which is augmented by a recombinant DNA molecule encoding 4-coumarate Co-enzyme A ligase, or a recombinant DNA molecule comprising an antisense 4-coumarate Co-enzyme A ligase gene, or fragment thereof, which plant has altered growth characteristics relative to the corresponding non-genetically altered plant.
Further provided is a method to genetically alter plants so as to change or alter their lignin structure. The method comprises introducing an expression cassette into cells of a plant, e.g., a tree, so as to yield genetically altered plant cells. The expression cassette preferably comprises an antisense recombinant DNA molecule, segment or sequence comprising a 4-coumarate Co-enzyme A ligase gene, or a fragment thereof. The transformed plant cells are regenerated to provide a regenerated, genetically altered plant. The recombinant DNA is expressed in the cells of the regenerated, genetically altered plant in an amount that alters the lignin structure in the cells of the plant relative to the corresponding non-genetically altered plant.
Also provided is a method for altering the lignin content in a plant. The method comprises introducing an expression cassette comprising a recombinant DNA molecule comprising a 4-coumarate Co-enzyme A ligase gene operably linked to a promoter functional in a plant cell into the cells of a plant. The plant cells are regenerated so as to yield a genetically altered plant. The recombinant DNA molecule is expressed in the cells of the regenerated plant in an amount effective to alter the lignin content in the plant cells. Preferably, the lignin content is reduced. Also preferably, the lignin content is reduced in a tissue-specific manner. In particular, a reduction in lignin content in forage crops is useful as the digestability of these crops by ruminants is increased. Also preferably, the 4-coumarate Co-enzyme A ligase gene is in an antisense orientation relative to the promoter.
Further provided is a genetically altered, e.g., transgenic, plant having an altered lignin content in the plant cells. The plant comprises a recombinant DNA molecule comprising a nucleotide sequence encoding a plant 4-coumarate Co-enzyme A ligase operably linked to a promoter so that the recombinant DNA molecule is expressed in an amount effective to alter the lignin content of the plant.
Yet another embodiment of the invention is a method to alter, e.g., increase, the cellulose content in plants. The method comprises introducing an expression cassette into cells of a plant, e.g., a tree, so as to yield genetically altered plant cells. The expression cassette preferably comprises an antisense recombinant DNA molecule, segment or sequence comprising a 4-coumarate Co-enzyme A ligase gene, or a fragment thereof, operably linked to a promoter functional in a plant cell. The transformed plant cells are regenerated to provide a regenerated, genetically altered plant. The recombinant DNA is expressed in the cells of the regenerated, genetically altered plant in an amount that alters the cellulose content in plant. Thus, the invention further provides a genetically altered, e.g., transgenic, plant having an altered cellulose content.
The invention also provides a method to genetically alter plants to increase their disease resistance, e.g., to fungal pathogens. The method comprises introducing an expression cassette comprising a recombinant DNA molecule comprising a nucleotide sequence encoding a 4-coumarate Co-enzyme A ligase operably linked to a promoter functional in a plant cell into cells of a plant. The transformed plant cells are regenerated to provide a genetically altered plant. The recombinant DNA molecule is expressed in the cells of the regenerated, genetically altered plant in an amount effective to render the plant resistant to disease. Preferably, the recombinant DNA molecule is expressed in amount that decreases the amount of lignin in the plant and/or increases the amount of phenolic compounds which are toxic to fungal pathogens. Hence, the invention also provides a transgenic plant, which is substantially resistant to disease. The plant comprises a native 4-coumarate Co-enzyme A ligase gene, and a recombinant DNA molecule comprising a nucleotide sequence encoding 4-coumarate Co-enzyme A ligase operably linked to a promoter functional in a plant wherein the recombinant DNA molecule is expressed in an amount effective to confer resistance to the transgenic plant.
Other features and advantages of the invention will become apparent to those of ordinary skill in the art upon review of the following drawings, detailed description and claims.